We are utilizing two approaches to identify and clone genes involved in the control of early development in the mouse. Homeobox genes. Embryonic patterning in Drosophila is controlled by a regulatory gene network. Many of the genes included in this network contain conserved sequence motifs such as the homeobox. The expression pattern of many of the murine homeobox (Hox) genes suggests that a similar, gene network may control development in this organism as well. However, the majority of the Hox genes that have been described are expressed relatively late during development (after gastrulation), and are probably not involved with very early determinative decisions. To investigate the role of homeobox genes in these early events, we have screened early embryonic cDNA libraries for homeobox genes that are specifically expressed during the very early stages of mouse embryogenesis (fertilization through early postimplantation development) using the polymerase chain reaction (PCR) technique and degenerate homeobox-specific oligonucleotides. We have cloned at least three new homeobox containing genes, and have shown by in situ hybridization that these genes are differentially expressed in the embryo during early postimplantation development. We have also identified a new homeobox gene family in the mouse with homology to the Distal-less gene of Drosophila, a gene necessary for development of the limbs of the fly. After determining the developmental expression profile for each gene, we plan to conduct expression studies in embryonic stem (ES) cells and transgenic mice to investigate their developmental functions. Insertional mutagenesis via enhancer trapping. To identify new genes involved in development we have introduced enhancerless beta-galactosidase (lacZ) constructs into ES cells. Expression is only detectable if the construct has integrated into an active gene region. We have screened for cells that show interesting patterns of lacZ expression in differentiating embryoid bodies and in chimeras, and have obtained several lines that show either activation or inactivation of expression as differentiation occurs. Breeding of chimeric mice obtained from these cells will potentially lead to the derivation of mouse mutants at these loci. The interrupted genes can easily be cloned by virtue of the molecularly distinguishable lacZ sequences. The significance of this work lies in the potential to study the largely inaccessible period of early mammalian embryogenesis on the molecular level. This will ultimately allow a better understanding of both normal and abnormal development.